Comparison of Two High-Pressure Liquid Chromatography Instruments Bio-Rad Variant-II and Tosoh HLC-723G11 in the Evaluation of Hemoglobinopathies

Abstract

Hemoglobin High-performance liquid chromatography (Hb HPLC) is a standard first-line technique for diagnosis of thalassemia and hemoglobinopathies. We compared two HPLC systems for detection and quantification of normal and abnormal Hb fractions. EDTA samples from 100 normal healthy subjects and 107 subjects affected with hemoglobinopathies or carriers were analysed using HPLC systems Tosoh HLC-723G11 and Bio-Rad Variant-II. Retention time (RT) and area of peaks for HbA2, HbF and other structural variants were compared. In discrepant cases samples were run on Sebia Capillary zone electrophoresis (CZE) for confirmation of results (39 out of 107 cases with HbE, HbD Iran, Hb Lepore and HbQ). Measurement of HbA2 and HbF in normal samples and HbF in those with variant Hbs showed good correlation by both analyzers (R ² = 0.83, 0.9 and 0.99 respectively). HbE co-elutes with HbA2 in Bio-Rad. Correlation done using the apparent HbA2 concentration from Bio-Rad with (HbE + HbA2) from Tosoh G11 showed good correlation (R ² = 0.97). Correlation of HbS (Eluting at S-window at RT 3.11 min in Tosoh G11 and 4.33 min in Bio-Rad) as well as HbD Punjab (Eluting at D-window at RT 2.82 min in Tosoh G11 and 4.06 min in Bio-Rad) by both instruments was good. HbD Iran (Eluting at E-window at RT 2.69 min in Tosoh G11 and with HbA2 at 3.53 min in Bio-Rad); HbQ (Eluting at C-window at RT 3.78 min in Tosoh G11 and unknown window at 4.7 min in Bio-Rad), HbH (Eluting at P00 window at RT 0.13 min in Tosoh G11 and giving pre-integration peak in Bio-Rad), Hb Lepore (Eluting at P08 window at RT 2.67 min in Tosoh G11 and with HbA2 at 3.46 min in Bio-Rad) gave comparable results. Correlation with findings of CZE was done in few cases when needed. Two automated HPLC instruments demonstrated similar usefulness for screening patients for hemoglobinopathies. However, complex elution patterns as well as co-elution of variants like HbA2, HbE, Hb Lepore, HbD Iran (in Bio-Rad); HbD Iran and HbE (Tosoh G11) pose difficulty in interpretation. A complementary second method like CZE may be required.

Keywords: Chromatogram; HPLC; Hemoglobinopathy.

Fig. 1

Regression analysis of results from Tosoh G11 versus Bio-Rad Variant II for a HbA2 in samples without Hb variants, b HbF in samples without Hb variants, c HbA2 in samples with variants, d HbE + HbA2 of Tosoh vs. HbA2 of Bio-Rad Variant II, e HbF in samples with variants, f HBS, and g HbD Punjab

Fig. 2

Chromatograms of HbE homozygous (a–c), HbE heterozygous (d–f) and E-beta (compound heterozygous) (g–i) on Bio-Rad Variant II, Tosoh G11 and Sebia CZE

Fig. 3

HPLC by Bio-Rad Variant II and Sebia CZE chromatogram showing unusual case of HbQ India with unknown alpha variant